Project Summary/Abstract Filopodia are finger-like extensions of the plasma membrane that allow cells to contact and interact with their surroundings in processes such as nerve growth, blood vessel formation, and the spread of cancer cells. A poorly understood structure at the tips of filopodia, the filopodial tip complex, constitutes a key site of filopodial actin polymerization, adhesion, and signaling. Our research with myosin-X (Myo10) shows that it is a major component of the filopodial tip complex and recently revealed that it has important roles in mammalian development in neural tube closure, eye formation, blood vessel development, and pigmentation. A patient with microphthalmia (unusually small eyes) was recently shown to lack Myo10, strongly suggesting that mutations in Myo10 can cause recessive genetic disease in humans. A growing number of papers also show that Myo10 is a key protein in the invasion, metastasis, and division of cancer cells and is frequently upregulated in major human cancers. These and other observations make Myo10 an attractive target for anticancer research and make it essential to determine the fundamental cell biological functions of Myo10 and filopodial tip complex. Although filopodial tips can form specialized sites of adhesion, they lack core components of focal adhesions. Virtually all filopodial tips contain Myo10, a protein that we have shown can link actin to ?-integrins, key molecules in cell adhesion. Because the assembly and composition of the tip complex and how it changes as it converts from extension to retraction or adhesion is not understood, we will: 1) Determine the composition of the filopodial tip complex during initiation, extension, retraction, and adhesion This aim will define the assembly pathway and states of the filopodia tip complex with respect to core filopodial tip components including Myo10, its putative interactor VASP, and capping protein. 2) Define the composition of the adhesions at the tips of mitotic retraction fibers. Although retraction fibers have the biologically crucial role of anchoring cells during mitosis, almost nothing is known about the composition of the adhesions at their tips. Myo10 is one of the few proteins known to localize to the tips of retraction fibers, so we will use it to investigate this poorly understood, but key site of cell adhesion. 3) Investigate the composition and functions of basolateral filopodia. We have discovered that in polarized epithelial cells Myo10 is targeted to the tips of filopodia on the basolateral surface rather than the apical microvilli on the same cells. Because Myo10 provides a probe for the tips of this largely uncharacterized class of filopodia, we will use it to investigate the basic cell biology of these structures. Together this research will define the basic properties of three poorly understood structure marked by Myo10 and the tip complex: filopodial tip adhesions, retraction fiber adhesions, and the filopodia on the basolateral surfaces of polarized epithelial cells.